Adolescents are more likely to initiate cigarette smoking than adults and adult smokers who initiated cigarette smoking during adolescence suffer from more severe nicotine addiction than adult-onset smokers. Recent data from a laboratory study in adult smokers and from a rat study using the reinstatement procedure (an animal model of drug relapse and craving) demonstrate that the response to smoking-associated cues in humans or intravenous nicotine-associated cues in adult rats progressively increases, or incubates during abstinence. Incubation of nicotine craving has not been previously assessed in rats exposed to nicotine during adolescence. In a preliminary study with rats that initiated nicotine self-administration during adolescence we found that cue-induced nicotine seeking in extinction tests was significantly higher after 28 withdrawal days than after 1 withdrawal day. The mechanisms underlying incubation of nicotine craving in rats that initiate nicotine self-administration as adolescents are unknown. In this exploratory grant, we propose behavioral and neurobiological studies to begin to characterize mechanisms underlying incubation of nicotine craving. At the behavioral level, we will determine the time course of incubation of nicotine craving and the impact of extended nicotine access on this incubation. At the neurobiological level, we will use a novel pharmacogenetic approach-the Daun02 inactivation method-to identify putative neuronal ensembles in specific brain areas that play a causal role in incubation of nicotine craving in adolescent-trained rats. We hope that our application will provide new insights on mechanisms underlying relapse to nicotine seeking in adolescent-onset adult smokers.